The detection of mutations in DNA is of importance in a variety of fields. One such field is the diagnosis of genetically determined diseases and to identify carriers of such diseases. It has been estimated that, in Northern Europe, diseases caused by genetically determined defects may affect 1% of all live births. In some Mediterranean countries, 20% of the population are said to have genetic defects, associated with thalassaemia.
Conventional methods of gene analysis involve DNA isolation and restriction digestion, gel electrophoresis and DNA blotting by the technique of Dr. E. Southern, hybridization and washing, and finally autoradiography. A total of 3-10 days are required and radioactive probes are used for hybridization. Such methods are the subject of a review by P. F. R. Little in "Genetic Engineering" volume 1, pages 61-102, published 1981 by Academic Press.
Such methods can be used whether or not the DNA has been accurately sequenced in the region of interest. But they have major disadvantages; they are only effective to detect point mutations where these happen to be present in a restriction enzyme cleavage site, and then only provided that there are not other nearby cleavage sites for the same enzyme; they require the tedious preliminary steps of DNA isolation, restriction, gel electrophoresis, and Southern blotting; and they generally require the use of radioactive labels. These disadvantages have inhibited the development of genetic screening in clinical laboratories by these techniques.
When the DNA sequence in the region of interest is known, it is possible to overcome some of these disadvantages. B. J. Conner et al (Proc. Natl. Acad. Sci. U.S.A., 80, January 1983, 278-282) describe a method which does not require the mutation to be at a restriction enzyme cleavage site. A radioactively-labelled 19-base oligonucleotide probe is caused to hybridize with the region of the DNA which includes the possible mutation. The hybridization conditions are carefully chosen so that the probe does or does not hybridize depending on whether the mutation is or is not present. But the length of the probe and the hybridization conditions are difficult to get right and are critical for success. The aforesaid tedious preliminary steps are used, as is a radioactively labelled probe.
The method of the present invention generally requires a knowledge of the nucleic acid sequence in the region of interest. But it does not require the mutation to be at a restriction enzyme cleavage site. (In the case of mutations within restriction sites, it may be possible to infer the base change involved from a simple end-filling experiment, without needing to know the exact nucleic acid sequence). The method is capable of giving unambiguous results. In the preferred forms, it does not require the tedious preliminary steps that characterize prior methods, and it may not require the use of a radioactive label.